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A novel ionization technique named medium vacuum chemical ionization (MVCI) mass spectrometry (MS), which is a chemical ionization using oxonium (H 3 O + ) and hydroxide (OH - ) formed from water, has excellent compatibility with the supercritical fluid extraction (SFE)/supercritical fluid chromatography (SFC). We have studied a method to determine free fatty acids (FFAs) in a small section of bovine liver tissue using SFE/SFC-MVCI MS analysis without further sample preparation. A series of FFA molecules interact with the C18 stationary phase, exhibiting broad chromatographic peaks when using a non-modified CO 2 as the mobile phase. It can be optimized by adding a small content of methanol to the mobile phase as a modifier; however, it may dampen the ionization efficiency of MVCI since the proton affinity of methanol is slightly higher than water. We have carefully evaluated the modifier content on the ion detection and column efficiencies. The obtained result showed that an optimized performance was in the range of 1 to 2% methanol-modified CO 2 mobile phase for both column efficiency and peak intensity. Higher methanol content than 2% degrades both peak intensity and column efficiency. Using optimized SFC conditions, a section of bovine liver tissue sliced for 14 µm thickness by 1 mm square, which is roughly estimated as about 3300 hepatocytes, was applied to determine 18 FFAs amounts for carbon chains of C12-C24. An amount of each tested FFA was estimated as in the range of 0.07 to 2.6 fmol per cell., (Copyright ©2024 Toshinobu Hondo, Yumi Miyake, and Michisato Toyoda.)

A method for the rapid determination of α -tocopherol ( α -T) and its oxidative products in plant tissue has been developed using supercritical fluid extraction (SFE) coupled with supercritical fluid chromatography (SFC) and medium vacuum chemical ionization (MVCI) with tandem mass spectrometry. The method is designed to study changes in levels for α -T and its oxidative products in plant cells during photosynthesis, aiming to observe the light response curves. α -T oxidation is a non-enzymatic self-defense mechanism in plant cells. Unlike enzyme-involved reactions, it cannot be stopped, so the oxidation continues in crude extracts even after extraction. Therefore, a real-time in-situ method is essential for tracking the light response curves. To optimize the selective reaction monitoring method, the reaction mixture of α -T and singlet oxygen ( 1 O 2 ), generated by rose Bengal under light illumination, was used as the source of oxidative products. The relative abundance changes in α -tocopherylquinone and 8a-hydroperoxy tocopherone in Pisum sativum L. (Pea) leaves under excessive light illumination have been preliminarily analyzed as part of the light response curve study. The method archives a throughput of 10-15 minutes for analyzing duplicate leaf samples. This process includes cutting off the leaf, sectioning it, placing the sample in a frozen SFE vessel, and conducting SFE/SFC analysis. Consequently, the average throughput is approximately 5-7 minutes per sample., (Copyright © 2024 Toshinobu Hondo, Yumi Miyake, and Michisato Toyoda.)